| The purpose of gene therapy, immune and metabolic regulation can be achieved byadministration of foreign plasmid DNA via the gastrointestinal tract (GI). The possibleinfluence of the absorption and gene transfer of foreign plasmid DNA on the organism hasdrawn considerable attention. Absorption of foreign plasmid DNA and effect of foreignplasmid DNA on immune function via the gastrointestinal tract were studied in this paper.Effect of foreign plasmid DNA on gene expression profile in vivo by DNA microarraytechnology platform was particularly investigated in the paper.1. Tissue distribution and evaluation of possible integration into host cellular DNA offoreign plasmid DNA following oral administration in miceThe absorption of foreign plasmid DNA in vivo and integration into host cellular DNAwere investigated in mice. KM mice, 5-6 weeks, 20±2g, were orally administered with 200μg plasmid pcDNA3s once. Lung, kidney, spleen, mesenteric lymph node, thymus, gonads,feces, duodenum, large intestine, blood, muscle and liver were obtained 1, 3, 6, 24 and 48 hand 3, 6 wk after oral administration in mice. Semiquantitative PCR technique was used todetect the distribution and kinetics of plasmid in different tissues after extracting total DNAfrom tissues. Genomic DNA can be assayed for integrated plasmid by PCR after purificationof high molecular weight genomic DNA away from free plasmid using gel electrophoresis.The sensitivity of PCR reaction was determined by PCR amplification of the control tissuegenome after administering different copies pcDNA3s plasmid. The plasmid clone in fecesbacterial in the intestinal was screened by ampicillin.The results showed that plasmid DNA was detected in almost all tissues 1, 3, 6, 24 and48 h and 3wk after oral administration of pcDNA3s plasmid. Foreign plasmid, however,was detected only in kidney at sixth week time. Foreign plasmid mainly survived asfragments in vivo. Each tissue genome was negative after PCR amplification and it was lowerthan the sensitivity of PCR reaction. No positive plasmid clone was detected in fecesbacterium, suggesting that plasmid DNA could not transform into bacterium in the intestine.In conclusion, foreign plasmid can be absorbed by gastrointestinal tract and distribute indifferent tissues quickly, surviving as the form of fragment in vivo. Foreign plasmid DNAcould not integrate into the host genome after oral administration.2. Studies on the mechanism of absorption of foreign plasmid DNA in the intestine. Six Balb/c mice, 5-6 weeks old, were randomly divided into two groups of 3 mice each.Control mice (n=3)were orally administered with 200 μL saline and the other group wereorally administered with 200 μg of plasmid pcDNA3 dissolved in 200 μL saline. Mice weresacrificed 4h after oral administration and jejunum was isolated from the mice. Total RNAwere extracted from the jejunum tissue. cDNA was synthesized by RT-PCR and was thentranscripted to cRNA. cRNA was reverse-transcripted again. The cDNA from the plasmidDNA group and the control group were labeled with Cy5 and Cy3, respectively. The labeledDNA were hybridized and cleaned with the gene chips. The signal was detected by scanningand data analysis was performed by GenePix Pro 4.0 software.The results showed that 4196 genes were expressed among the total 17667 genes. A totalof 61 genes showed significant change in their expression level after plasmid pcDNA3administration, among which 36 genes were up-regulated and 25 were down-regulated. Theexpression of intestinal anion transport protein was induced after oral administration offoreign plasmid DNA, suggesting that plasmid DNA absorption mediated by transport proteinwas occured in the intestine. Foreign plasmid DNA could cause differential expression inimmune response genes in the intestine. Foreign plasmid DNA could improve intestinalanti-oxidation function by up-regulating the expression of microsomal glutathioneS-transferase and glutathione peroxidase. Foreign plasmid DNA inhibited apoptosis inintestinal mucosa by down-regulating Caspase3. Mitogen activated protein kinase 2 wasup-regulated in the intestine, indicating that foreign plasmid DNA could activate intestinalmucosa cell through MAPK pathway.3. Effect of oral administration of foreign plasmid DNA on immune function in miceEffect of foreign plasmid on immune function of normal and immunodepression micevia the gastrointestinal tract was studied. After oral administration of foreign plasmidpcDNA3, thymus and spleen index, anti-sheep red blood cell (SRBC), number of antibodysecreting cell (NASC) in spleen and phagocytic activity were detected. lymphocytictransformation rate (LTR) in spleen was determined using MTT methods. Serum IgA, IgGand IgM in immune-suppressed mice were also examined with immunoglobulin kit. Thymusand spleen index, LTR, anti-SRBC and NASC in spleen were significantly increased afteradministration of foreign plasmid pcDNA3 (3.53±0.80 vs 5.10±0.47 mg/g, P <0.01;5.69±0.92 vs 7.49±1.18 mg/g, P <0.01;1.047±0.012 vs 1.154±0.016, P <0.01;6.46±0.12 vs8.18±0.29, P <0.01;0.403±0.008 vs 0.471±0.007, P <0.01;respectively). Phagocytic activityalso increased significantly (phagocytic index: 0.53±0.017 vs 0.72±0.029, P <0.01);(phagocytic ratio: 32.30±1.098 vs 60.53±2.022, P <0.01). The serum IgA, IgG and IgM ofimmune-suppressed mice resumed to normal level. All these results indicated that foreignplasmid DNA might induce humoral and cell mediated immune in mice after administered viathe gastrointestinal tract.4. Studies on the molecular mechanism of immune response induced by foreign plasmidDNATo investigate the molecular mechanism of foreign plasmid DNA-induced immuneresponse in vivo, spleen gene expression profile was studied by microarray after oraladministration of plasmid DNA. Nine Male Balb/c mice were randomly divided into threegroups of 3 each. Control group were orally administered with 200μl stroke-physiologicalsaline solution. Another two groups were orally administered with 200 μg of plasmidpcDNA3 in 200 μL saline once and were sacrificed at 4h , 18h after orally administration.Spleen was excised and total RNA was extracted. Affymetrix array process included cDNAsynthesis and purification, cRNA synthesis and purification, microarray hybridizationscanning and data analysis. Functional cluster analysis was conducted in all physiologicalprocess including biological process, molecular function and cellular component by Genmappsoftware.The results showed that 1692 genes were up-regulated and 502 genes weredown-regulated, among which 276 genes were significantly up-regulated (SLR≥1) and 447significantly down-regulated (SLR≤-1) at 4h after oral administration, while 1456 geneswere up-regulated and 123 genes were down-regulated, among which 395 genes weresignificantly up-regulated (SLR≥1) and 70 significantly down-regulated (SLR≤-1) at 18hafter oral administration. Up-regulated expression was observed in a lot of genes participatedin immune response, including cytokine, chemokine, cytokine and chemokine receptors,major histocompatibility antigens, complement molecular, lymphocyte differentiation antigen,proteasome and apoptosis genes. The immunoglobulin genes were down-regulated.MAPPFinder result also showed that plenty of immune response processes were up-regulated.These results indicated that foreign plasmid DNA can modulate the expression of immunegene in spleen after oral administration.5. Effect of foreign plasmid DNA on substance and energy metabolism in spleen.The differentially expressed genes were further analyzed by Genmapp based on spleenmicroarray result. Genmapp result showed that a lot of metabolic pathway were up-regulatedwhen immune response was induced in spleen after oral administration of plasmid. Thesemetabolic pathway included pyrimidine and purine synthesize , protein synthesis, cholesterolsynthesis, fatty acid synthesis, glycolysis , TCA cycle and mitochondria oxidativephosphorylation. Immune cell proliferated generously when stimulated by foreign plasmidDNA. The acceleration of the process of DNA replication could induced synthesis ofpyrimidine and purine and accordingly provided conditions for the up-regulation of synthesisof protein and lipid. And the up-regulation of oxidative phosphorylation , glycolysis,gluconeogenesis and TCA cycle may provide essential energy for cell division andproliferation.6. Effect of foreign plasmid DNA on acute phase response in the liverThe method was the same as part 5. Liver was isolated and total RNA was extractedfrom liver for subsequent microarray. The result showed that 3400 genes expressed among thetotal of 17664 genes. 141 genes showed significant expression, among which 100 genes wereup-regulated and 41 genes were down-regulated. The up-regulated genes mainly participatedin immune response, cell cycle, development, transcription factors, signal transduction,transport and metabolism;while the down-regulated genes mainly participated in metabolismand transport.Some hepatic acute phase response protein were also up-regulated, such as serumamyloid A protein, haptoglobin, insulin-like growth factor binding protein 1, metallothionein,etc. Serum amyloid A protein 3 was significantly up-regulated (fold=30.7). Interleukin 1 andinterleukin 17 receptors were also induced, which modulate the acute phase response in theliver. The biological process changes in liver are mainly involved in the potentiation of acutephase response, the activation of immune response and cellular signal pathway and thesuppression of lipid synthesis after oral administration of plasmid DNA.In conclusion, foreign plasmid DNA can be absorbed via the gastrointestinal tract.Foreign plasmid DNA may modulate immune response and metabolic pathway in vivo afterabsorbed by the gastrointestinal tract. High throughput microarray may be a valid method forthe investigation of effect of foreign plasmid DNA on gene expression profile in vivo, andfurther analysis of related genes and process may help understand the mechanism of action offoreign plasmid DNA absorbed via the gastrointestinal tract.
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